To more completely assess the fungal species to which people are exposed, fungal Internal Transcribed Spacer (ITS) region gene sequencing studies were designed to test the hypothesis that the assemblage of fungal bioaerosols in the United States built and occupational environments are much richer than previously estimated using traditional methods of analysis. Fungal ITS sequencing data from samples collected from a NIOSH Elementary School Study, a pre and post remediated office building in the northeastern United States, as well as air and dust samples derived from NIOSH Health Hazard Evaluations have been analyzed using ITS region sequencing to characterize the diversity of fungal communities in air and dust samples. The results of ITS region sequencing studies have provided insight into the contribution of previously overlooked fungi in various occupational environments. For example, ITS region sequencing of dust samples (n=500) collected from 50 elementary schools with staff reporting respiratory symptoms identified the most abundant fungi to include allergenically important species placed in the order Pleosporales, Phoma, Epicoccum, Alternaria. A recent ITS region sequencing analysis of samples from an office building in the northeastern United States that had known water incursion before (n=27) and after (n=27) building remediation has shown unicellular yeasts to be among the most abundant fungi in floor dust samples. Other plant-associated fungi belonging to the orders Pleosporales and Capnodiales were also frequently detected in the ITS sequence analysis. Interestingly, post-remediation, many of the same fungi and yeast species remained abundant. In an analysis of area and personal air samples collected in occupational environments such as the emerging cannabis production industry has identified the spectrum of fungi to be composed of pathogenic plant species including Botrytis cinerea and Wallemia sebi. Spores derived from these species were shown to be aerosolized into the breathing zone of workers conducting occupational job tasks such as handling and trimming cannabis. Ongoing studies are also examining the assemblage of fungal species present in the New York City Neighborhood Asthma and Allergy Study led by scientists located at Columbia University and the Ohio State University. Research performed by the collaborative study group has shown dust samples to contain fungal taxa including Aureobasidium pullulans, Penicillium glabrum, W. sebi and Alternaria alternata. These prevalent fungal taxa varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. Additional studies suggest that multiple environmental factors including anthropogenic behavior modification, housing type, and neighborhood are important variables that influence fungal community profiles within middle-income homes in New York City. Unicellular yeasts placed in the genus Cryptococcus have recently been shown to be among the most frequently detected genera in the ITS region sequencing analysis. A quantitative polymerase chain reaction method was recently developed at NIOSH and has been employed in an ongoing evaluation of the Neighborhood Asthma and Allergy Study samples.